Specificity in the detection of anti-rubella IgM antibodies

ABSTRACT

This invention particularly discloses an improved immunoassay method for the specific detection of anti-rubella IgM antibodies. This method employs rubella antigens comprising rubella E1 and E2 envelope glycoproteins substantially free of capsid protein. Use of this antigen composition reduces or eliminates nonspecific protein-protein interactions leading to false positive results. A sample diluent comprising urea can also be used in the process to further reduce the occurrence of nonspecific protein-protein interactions. A diagnostic kit to aid in the detection of anti-rubella IgM antibodies is also disclosed.

This is a continuation of application Ser. No. 09/203,161, filed Dec. 1,1998 now abandoned.

FIELD OF THE INVENTION

Improved methods for the specific detection of anti-rubella IgMantibodies in biological samples are disclosed.

BACKGROUND OF THE INVENTION

The rubella virion is a member of the Togavirus family, and is aspherical, enveloped virus approximately 60 nm in diameter. The virionconsists of a 10 kb single-stranded RNA molecule encapsidated in anicosahedral nucleocapsid and surrounded by a lipid envelope. Multiplecopies of a capsid (C) protein make up the nucleocapsid. The envelopeconsists of lipoproteins derived from the infected host cell and twoviral glycoproteins, designated E1 (53-58 kDa) and E2 (42-48 kDa)(Waxham and Wolinsky, Virology 143:153-165, 1985).

Primary rubella infection is characterized in most individuals by thepresence of a macropapular rash, fever, malaise, and lymphadenopathy.Rubella is typically a mild and self-limited disease, and is most oftencontracted during childhood. Primary infection in adults is less common,and may have very serious consequences in pregnant women. Infection of afetus during the first trimester of pregnancy may result in spontaneousabortion or severe fetal abnormalities. A congenitally infected infantmay exhibit one or more of a variety of birth defects collectively knownas congenital rubella syndrome (CRS). Common birth defects associatedwith CRS include cataracts, central nervous system deficits,microcephaly, motor deficits, deafness, congenital heart disease, andmental retardation.

Because of the fetal risk associated with primary maternal rubellainfection, test samples from pregnant women are routinely screened forthe presence of anti-rubella IgM antibodies during the first trimesterof pregnancy. Primary rubella infection is associated with a pronouncedspecific IgM antibody response, while reinfection (often asymptomatic)is characterized by elevated levels of specific IgG antibodies in theabsence of detectable levels of specific IgM antibodies. Unlike primaryinfection, reinfection of immune pregnant women is generally thought tobe harmless to the developing fetus. When prenatal screening indicatesthat a woman has acquired a primary rubella infection during the earlystages of pregnancy, a therapeutic abortion is often recommended. As aresult, it is imperative that the test results are accurate.

A variety of methods related to the detection of anti-rubella IgMantibodies have been described. Initial assays relied onhemagglutination inhibition (HAI) testing, which is dependent upon thehemagglutinating properties of the viral E1 and E2 glycoproteins. If abiological sample to be tested contains antibodies directed againstthese viral hemagglutinins, rubella virus can no longer bind to redblood cells (usually from chicken blood) and this inhibitshemagglutination (see e.g. Peetennans and Huygelen, Presse Med.75:2177-2178, 1967). Unfortunately, titers of these anti-hemagglutininantibodies increase significantly following both primary infection andreinfection, and so this method cannot be used to distinguish betweenmaternal infections likely to result in fetal defects and maternalinfections unlikely to affect the fetus.

More recently, enzyme-linked immunosorbent assays (ELISAs) have becomethe method of choice in the diagnosis of primary rubella infection (seee.g. Steece et al., J. Clin. Microbiol. 21(1):140-142, 1985). In mostcases, rubella viral capsid or envelope glycoprotein antigens (wholeproteins, viral extracts, or peptides) are immobilized on a solidsupport and exposed to a biological sample to be tested for the presenceof anti-rubella antibodies. Antigens previously described include novellinear and cyclic peptides corresponding to regions of the rubella E1and C proteins (U.S. Pat. Nos. 5,164,481 and 5,298,596), novel linearand cyclic peptides corresponding to regions of the E1 and E2glycoproteins (U.S. Pat. No. 5,427,792) and intact rubella virus (orantigens or fragments thereof) in which oligosaccharide moieties havebeen modified for better recognition by antibodies (U.S. Pat. No.4,965,069). Any anti-rubella antibodies present in the test sample bindto the immobilized antigen. After appropriate washing, the presence orabsence of bound antibody is detected through the use of an indicatorreagent capable of complexing with an anti-rubella IgM antibody. Thisindicator reagent is typically conjugated to a detectable label. Afterwashing, the bound detectable label is quantified directly orindirectly, and the result is used to determine whether or not theinitial sample contained specific anti-rubella IgM antibodies. Thepresence of specific anti-rubella IgM antibodies in a sample forms thebasis for a diagnosis of primary rubella infection.

Unfortunately, there is a disturbing lack of specificity in existinganti-rubella IgM immunoassays. False positive results are obtained sofrequently that most laboratories use a complicated testing algorithm toconfirm an occurrence of primary rubella infection. This algorithmincludes repeated testing of each sample employing differentanti-rubella IgM diagnostic kits, a time-consuming and expensiveprocess. The algorithm may also include complementary testing forspecific anti-rubella IgG antibody avidity. The initial immune responsefollowing exposure to a novel antigen is characterized by the productionof an abundance of IgM antibodies, whereas the specific high-avidity IgGantibody response is characteristic of secondary responses andreinfection. Consequently, if IgG antibodies from a patient sample takenduring early pregnancy bind to rubella antigens with high avidity, it isunlikely that the initial maternal infection occurred during thecritical early stages of fetal development. Conversely, if the IgGantibodies in the patient sample bind the antigen with low avidity, itis suggestive of a recent primary infection with the rubella virus.

Several potential causative factors have been implicated in the lack ofspecificity in anti-rubella IgM immunoassays, and attempts havepreviously been made to reduce the incidence of false positive assayresults. For example, Macioszek et al. (U.S. Pat. No. 5,698,393, issuedDec. 16, 1997; “Macioszek”) disclose a method for reducing oreliminating false positive IgM immunoassay results caused by thepresence of rheumatoid factors (autoantibodies against human IgG,usually of the IgM isotype) in tested samples. This method involvestreatment of samples suspected of containing rheumatoid factors (RF),either directly or while in complex bound to the solid phase, with a RFneutralization citrate buffer of a pH most preferably between 3.5 and5.0. Macioszek teaches that the binding of nonspecific IgM moleculessuch as RFs to anti-rubella IgG molecules captured by the affixedantigen is typically disrupted within this pH range, while binding ofspecific IgM antibodies to immobilized rubella antigen is not disrupted.This patent does not address the problem of assay nonspecificityresulting from the presence of causative factors other than RF, and RFare only present in a subset of tested sera.

Fabrizi et al. (U.S. Pat. No. 5,300,427, issued Apr. 5, 1994; “Fabrizi”)also disclose a method for reducing nonspecific signal generated in IgMELISAs. Examples include an assay for IgM molecules recognizing“extractive antigens” of rubella. This method comprises dilution ofserum with buffer containing type IV collagenase prior to placing theserum in contact with the solid support, upon which is affixed anti-IgMantibodies. The complement system is composed of a set of plasmaproteins that attack extracellular pathogens. One of these complementproteins, C1q, which is capable of binding serum IgM antibodies,allegedly interacts nonspecifically with the enzyme-conjugated secondaryantibody. Fabrizi teaches that collagenase aids in the separation ofserum IgM molecules from attached C1q complement molecules, and therebyimproves the specificity of the immunoassay by eliminating falsepositive signals resulting from the abovementioned nonspecificinteraction. This patent does not address the problem of assaynonspecificity resulting from the presence of factors other thancrossreactive C1q protein in the biological sample.

There exists a need for an improved immunoassay for the detection ofanti-rubella IgM antibodies which eliminates all or most of the falsepositive results generated by current assays without affecting thesensitivity of the assay.

SUMMARY OF THE INVENTION

In a preferred embodiment, the present invention provides improvedimmunoassays for the detection of anti-rubella IgM antibodies. Theseassays comprise contacting a test sample suspected of containinganti-rubella IgM antibodies with rubella antigens comprising rubella E1and E2 glycoproteins substantially free of rubella capsid protein. Thepresent inventor has found that rubella capsid protein may be involvedin nonspecific protein-protein interactions leading to false positiveassay results.

These assays may further comprise the use of urea in sample diluent.Urea incorporation may further reduce nonspecific binding of IgMmolecules to the rubella E1 and E2 glycoproteins used as antigen in theimproved immunoassay, thereby farther reducing the incidence of falsepositive assay results.

In additional preferred embodiments, kits and compositions are providedto facilitate performance of the disclosed immunoassays.

DEFINITIONS

The following definitions are provided in order to aid those skilled inthe art in understanding the detailed description of the presentinvention.

“Agglutination” refers to the clumping together of particles, usually byantibody molecules binding to antigens on the surfaces of adjacentparticles.

“Antibody” refers to a protein which binds specifically to a givenantigen and is produced in response to initial contact with thatantigen.

“Antigen” refers to a molecule or discrete segment thereof capable ofeliciting an immune response, wherein the immune response results in theproduction of antibodies reactive with the antigenic epitope.

“Avidity” refers to the sum total of the strength of binding of twomolecules to each other.

“Binary complex” refers to a complex of antigen and IgM, for example, a)the rubella E1 glycoprotein and an anti-rubella IgM antibodyspecifically recognizing the E1 glycoprotein; b) the rubella E2glycoprotein and an anti-rubella IgM antibody specifically recognizingthe E2 glycoprotein; or c) a combination of E1 and E2 and IgM.

“Cloud point” refers to the temperature at which sudden turbidityappears during warming of a clear micellar detergent solution, theresult of a microscopic phase separation.

“Detectable label” refers to molecule, protein, or nucleic acid whichmay be detected either directly or indirectly through the use of asuitable detection agent or detection device.

“Detection agent” refers to a composition providing conditions suitablefor detecting a detectable label. Such compositions often allow theobservation of a colorimetric, fluorescent, or chemiluminescent signalwhen the detectable label is contacted with the detection agent.

“IgM” refers to the antibody isotype characteristic of the primaryantibody response following initial exposure to a given antigen.

“IgG” refers to the dominant serum immunoglobulin isotype, which ischaracteristically produced in secondary phases of the initial infectionand in subsequent reinfections.

“Indicator reagent” refers to a molecule, protein, or nucleic acidcapable of complexing with an anti-rubella IgM antibody. The bindingcomponent may be conjugated to a detectable label.

“Hemagglutination” refers to agglutination of red blood cells.

“Rubella E1 and E2 envelope glycoproteins substantially free of rubellacapsid protein” refers to a preparation of rubella envelopeglycoproteins, in which no capsid protein is detectable when thepreparation is subjected to SDS-PAGE followed by silver staining.

“Ternary complex” refers to a complex of antigen, IgM and indicatorreagent, for example, a) the E1 glycoprotein, an anti-rubella IgMantibody specifically recognizing the E1 glycoprotein, and an indicatorreagent; b) the E2 glycoprotein, an anti-rubella IgM antibodyspecifically recognizing the E2 glycoprotein, and an indicator reagent;or c) E1 and E2 in combination with IgM and an indicator reagent.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Biological samples to be tested by the instant assay are contacted withrubella E1 and E2 envelope glycoproteins substantially free of rubellacapsid protein, as Western blots performed by the instant inventorssuggest that some of the false positive results generated in otherassays may result from nonspecific binding of IgM molecules to therubella capsid protein. The assays of the instant invention may furthercomprise addition of urea to sample diluent, in order to further reducethe incidence of nonspecific protein-protein interactions leading tofalse positive assay results. The method of the instant improvedimmunoassay typically comprises several steps, as outlined below.

Formation of a Binary Complex

A test sample suspected of containing anti-rubella IgM antibodies isprovided. The test sample is contacted with rubella antigens comprisingrubella E1 and E2 envelope glycoproteins substantially free of rubellacapsid protein. This results in the formation of a binary complexcomprising a) the E1 glycoprotein and an anti-rubella IgM antibodyspecific for the E1 glycoprotein; b) the E2 glycoprotein and ananti-rubella IgM antibody specific for the E2 glycoprotein; or c) acombination of E1 and E2 bound to IgM. The binary complex can be washedseveral times to effectively remove any uncomplexed material.

Formation of a Labeled Ternary Complex

The binary complex is then contacted with an indicator reagent. Theindicator reagent typically comprises a binding molecule capable ofcomplexing with an anti-rubella IgM antibody. This results in theformation of a ternary complex comprising a) the E1 glycoprotein, ananti-rubella IgM antibody specific for the E1 glycoprotein, and theindicator reagent; b) the E2 glycoprotein, an anti-rubella IgM antibodyspecific for the E2 glycoprotein, and the indicator reagent; or c) E1and E2 bound to IgM and indicator reagent. The indicator reagent isgenerally conjugated to a detectable label. This ternary complex can bewashed several times to remove any uncomplexed material. The ternarycomplex will preferably be washed prior to the detection step.

Formation of a Labeled Ternary Complex in a Single Step

Alternatively, the test sample can be contacted with rubella E1 and E2envelope glycoproteins substantially free of rubella capsid protein andthe indicator reagent in a single simultaneous step. A ternary complex(as described above) may form. A complex formed in this manner can bewashed to remove uncomplexed material. The ternary complex willpreferably be washed prior to the detection step.

Detection and Diagnosis

The ternary complexes can be detected either directly or with a suitabledetection agent. The particular detection agent selected will depend onthe type of detectable label used. A positive signal indicates thepresence of anti-rubella IgM antibodies in the test sample, therebysuggesting that the individual providing the test sample may haverecently acquired a primary rubella infection. Conversely, the absenceof a signal indicates the absence of anti-rubella IgM antibodies in thetest sample.

The organism providing the test sample is generally any organism whichcontains antibodies. The organism preferably is a mammal, and morepreferably is a human.

The test sample can generally be any biological material containingantibodies. Such materials can be processed so that they are provided ina suitable form. The test sample is preferably provided from a bodilyfluid, more preferably is provided from blood, and most preferably isprovided from serum.

The test sample can be diluted with a sample diluent, which can compriseurea (N₂H₄CO). The incorporation of urea, a compound with chaotropicactivity, reduces the nonspecific binding of IgM to the abovementionedE1 and E2 envelope glycoproteins and/or a solid phase support materialused in the assay. The urea increases the stringency of theantibody:antigen interaction, with the effect that low-avidity complexesresulting in false positive assay results are excluded. Such low aviditycomplexes can be composed of nonspecific IgM molecules that have boundto cross-reactive epitopes on the rubella E1 or E2 glycoproteins.Preferably, the sample diluent will comprise between about 1 M and about5 M urea, more preferably between about 2 M and 4 M urea, and mostpreferably about 3 M urea.

The wash buffer used to wash the bound binary and/or ternary complexesdescribed above can comprise urea. The wash buffer will preferablycomprise about 1 M urea to about 5 M urea. More preferably, the washbuffer will comprise about 2 M urea to about 4 M urea, and mostpreferably about 3 M urea. One of ordinary skill in the art is awarethat these wash buffers may otherwise vary in their composition, butstill be compatible with the present invention.

The rubella E1 and E2 envelope glycoproteins substantially free ofrubella capsid protein can be prepared from any strain of rubella virus,including the Therien, Judith, M33 and HPV₇₇ strains. These E1 and E2envelope glycoproteins can be purified from capsid protein by any methodcompatible with the assay of the instant invention. Preferably, theseglycoproteins will be purified away from capsid protein through phaseseparation into detergent micelles, and most preferably, theseglycoproteins will be purified according to the method described inExample 1. It will be recognized by those of skill in the art thatchanges can be made in the specific method described while stillobtaining a like or similar result and without departing from the spiritand scope of the disclosed method. Alternatively, rubella E1 and E2glycoproteins substantially free of rubella capsid protein can also beobtained by other methods well known to those of skill in the art,including alternative purification methodologies, translation in invitro translation systems, and expression in bacteria, yeast, CHO cells,insect cells, or other cell systems. All such variations are consideredequivalent for the purposes of the present invention.

The E1 and E2 viral glycoproteins substantially free of rubella capsidprotein can be immobilized on a solid support. The solid support can beprovided in one of many different forms. Representative examples ofsolid support materials include membranes, filters, glass, plastic,plastic beads, agarose beads, SEPHAROSE® (polysaceharide) beads(SEPHAROSE® is a registered trademark of Pharmacia Biotech, Piscataway,N.J.), and magnetic beads.

In addition to the different forms, the solid support can be composed ofa variety of materials. The solid support is preferably nitrocellulose,polyvinylidene difluoride, nylon, rayon, cellulose acetate, agarose,SEPHAROSE® (polysaccharide) beads, metal, polypropylene, polyethylene,polystyrene, polyvinyl chloride, polyvinyl acetate, polyamide,polyimide, polycarbonate, polyether, polyester, polysulfone, polyacetal,polystyrene, or polymethyl methacrylate; more preferably ispolypropylene, polystyrene, polyvinyl chloride, polyamide,polycarbonate, polyether, polymethyl methacrylate, nitrocellulose,polyvinylidene difluoride, or nylon; and most preferably ispolypropylene or polystyrene.

The indicator reagent is typically conjugated to a detectable label. Thedetectable label can be an enzyme, such as alkaline phosphatase,β-galactosidase, or peroxidase; a protein, such as biotin or digoxin; afluorochrome, such as rhodamine, phycoerythrin, or fluorescein; afluorescent protein such as GFP or one of its many modified forms; aradioisotope; or a nucleic acid segment.

Some of these detectable labels can be detected directly. Fluorochromes(Wells and Johnson. In Three-Dimensional Confocal Microscopy, pp.101-129, 1994) and fluorescent proteins (Sakai et al., J. Cell Biol.139(6):1465-1476, 1997) can be directly detected with a suitabledetection device, such as a fluorescent microscope, fluorescentactivated cell sorter (FACS), or fluorometer. Radioisotopes (such as ³²Por ¹²⁵I) can be detected through the use of a scintillation counter orGeiger counter.

Other labels can be detected indirectly. These labels may require theuse of a suitable detection agent. The choice of a suitable detectionagent generally depends on which detectable label is used.

For example, if a protein such as biotin is used as the detectablelabel, a detection agent comprising avidin or streptavidin is generallyemployed (Bayer et al., Meth. Biochem. Anal. 26: 1-10, 1980). In suchcases, a suitable detection agent generally comprises a bindingcomponent capable of complexing with the protein. This binding componentcan be further detected by contacting it with a second detectable label.

Enzymes, such as horseradish peroxidase, alkaline phosphatase, andβ-galactosidase, can also be used as detectable labels. Detection agentsfor enzymes generally utilize a form of the enzyme's substrate. Thesubstrate is typically modified, or provided under a set of conditions,such that a chemiluminescent, calorimetric, or fluorescent signal isobserved after the enzyme and substrate have been contacted (Vargas etal., Anal. Biochem. 209: 323, 1993).

Nucleic acids, when used as detectable labels, can be detected throughthe use of a hybridizing probe. This probe can be conjugated to anadditional detectable label which, when placed under suitableconditions, provides a fluorescent, chemiluminescent, calorimetric, orradioactive signal. Alternatively, the nucleic acid can be amplified bymeans of a polymerase chain reaction (PCR) (Dirks et al., J. Histochem.Cytochem. 38:467-476, 1990). The amplified nucleic acid can be easilydetected by gel electrophoresis.

Radioisotopes can alternatively be detected indirectly byautoradiography (i.e. exposure to x-ray film).

There are many other suitable detection methods compatible with theinstant invention. In each case, the detection agent and its method ofuse are well known to one of ordinary skill in the art.

A diagnostic kit can be designed to aid in the performance of the abovemethod. Such a kit can contain vessels containing rubella E1 and E2envelope glycoproteins substantially free of rubella capsid protein andthe indicator reagent, respectively. The kit can further contain testsample diluent, various blocking buffers, buffers to aid the formationof binary and ternary complexes, wash buffers, and detection agents. Oneof ordinary skill in the art is aware that the diluent and buffers canvary in their exact composition, but still be compatible with thepresent invention. All such variations are considered equivalent for thepurposes of the present invention. The test sample diluent and/or washbuffers can comprise urea, as described above.

The rubella E1 and E2 envelope glycoproteins substantially free ofrubella capsid protein supplied in the diagnostic kit can be purified asdescribed above from any strain of rubella virus.

The E1 and E2 viral glycoproteins substantially free of rubella capsidprotein supplied in the diagnostic kit can be provided alreadyimmobilized on a solid support. This solid support can be provided inone of many different forms, and be composed of a variety of materials,as described above.

The diagnostic kit can further comprise a detection agent. As previouslymentioned, the choice of a suitable detection agent generally depends onwhich detectable label is used. Many different detectable labels anddetection agents are compatible with the present invention.

The components of the diagnostic kit can be provided in many differentforms and quantities. Various types of packaging can also be used.Instructions for the correct use of the kit can be supplied with thekit. Any such alternative embodiments are considered equivalent to thepresent invention.

In a further preferred embodiment, the invention relates to acomposition comprising rubella E1 and E2 envelope glycoproteinssubstantially free of rubella capsid protein, as described above.

In yet another preferred embodiment, the invention relates to a solidsupport material having immobilized thereon rubella E1 and E2 envelopeglycoproteins substantially free of rubella capsid protein. The rubellaE1 and E2 glycoproteins substantially free of rubella capsid protein canbe prepared according to any of the methods described above, and can beaffixed to any of the solid support materials previously described.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Preparation of Rubella Virus Glycoproteins

Partially purified or highly purified rubella virus in buffer containing150 mM NaCl, 10 mM Tris-HCl (pH 8.15), 10 mM EDTA, and 1.0 M KCl(NTE/KCl) was adjusted to 0.1 to 1.0 mg/mL protein by dilution in thesame NTE/KCl buffer. TRITON™ X-114 surfactant (TRITON™ is a registeredtrademark of Rohm and Hans Co., Spring House, Pa.) was then added to afinal concentration of 1% (vol/vol). The solution was placed in anice-water bath for 30 minutes and mixed at intervals by vortexing, afterwhich it was allowed to incubate at room temperature for 10 to 15minutes. As the solution warmed, it became turbid due to a microscopicphase separation that occurred when the solution temperature passed thecloud point. During the room temperature incubation the solution wasmixed vigorously two times by vortexing for 30 to 60 seconds to maximizethe interaction of the glycoproteins with the forming micelles. Thesolution was then centrifuged for 10 minutes at 1000 ×g at roomtemperature to collect the detergent phase at the bottom of the tube.The upper aqueous phase, containing the capsid protein, was removed andthe detergent phase was solubilized in the original volume of coldNTE/KCl. After a few minutes in the ice-water bath, the temperature ofthe solubilized detergent phase was allowed to rise above the cloudpoint with mixing, thereby allowing phase separation to occur asdescribed above. The detergent phase was collected and solubilized andthe phase separation was repeated twice. After the third phaseseparation, the detergent phase containing the glycoproteins wassolubilized in one-half the original volume of cold NTE buffer. Thedetergent was removed by three extractions with cold ether, after whichthe rubella virus glycoproteins were in the aqueous phase. Capsidprotein was not detectable as an impurity in the extractedglycoproteins, as demonstrated by SDS-PAGE followed by silver staining.

Example 2 Preparation of the Solid Support Material

The rubella virus glycoproteins were assayed in an antigen-dilution IgMELISA to determine the optimal antigen concentration for sensitizationof the solid phase. The glycoprotein solution was diluted inbicarbonate-carbonate buffer, pH 9.5, to within the range of 0.1 mcg/mlto 10 mcg/ml, typically 1 mcg/ml, and 0.1 mL was placed in each well ofa 96-well microtiter plate. The plates were incubated at 4-7° C.overnight to allow the glycoproteins to adsorb to the plastic surface.After adsorption, the fluid was removed from the plate wells. A blockingsolution of phosphate buffered saline, pH 7.0 containing 1% w/v bovineserum albumin and 5% w/v sucrose (0.2 mL) was then added to each welland the plates were incubated overnight at 4-7° C. The blocking solutioncontained proteins that adsorb to the remaining sites on the plasticwells and help block subsequent adsorption of the test serum globulinsto the plastic surface. After blocking, the fluid was removed from theplate wells and the plates were allowed to air dry. The plates were thensealed in a foil pouch along with a dessicant bag.

Example 3 Preparation of the IgM Serum Diluent

The serum diluent for the IgM ELISA consisted of phosphate bufferedsaline containing 1 M KCl, goat antihuman IgG (Fc specific) and 3 Murea. The goat antihuman IgG obtained from DiaSorin, Stillwater,Minnesota, was first cross species adsorbed with bovine gamma globulin,rabbit gamma globulin, etc. and was adjusted in concentration toprecipitate human IgG in a range from 5 to 20 mg/mL.

Example 4 Method for Performing an IgM ELISA

The serum test samples, an IgM calibrator serum and an IgM positivecontrol serum were individually diluted 1:10 or greater in the IgM serumdiluent of Example 3. The dilutions were allowed to incubate at roomtemperature for 10 to 60 minutes, after which 0.1 mL of each dilutedserum was placed in a separate well of the antigen-coated platedescribed in Example 2. After addition of all the serum samples, theplate was incubated in a moist chamber at room temperature for 30minutes. The fluid was removed by inverting the plate over a sink orbeaker and then slapping the plate on paper towels to remove any excessdiluted serum. Each well was washed three times with a wash bufferconsisting of phosphate buffered saline containing 0.1% (wt/vol) bovineserum albumin and 0.05% (vol/vol) TWEEN™-20 detergent (TWEEN™ is aregistered trademark of Robin and Haas Co., Spring House, Pa.). Thewells were filled with wash buffer and the fluid was removed asdescribed above. After the final wash was removed from the wells, 0.1 mLof goat antihuman IgM conjugated with alkaline phosphatase obtained fromKirkegaard and Perry Laboratories, Inc., Gaithersburg, Md. (IgMconjugate) was placed in each well. The IgM conjugate was diluted inphosphate buffered saline containing 1% (wt/vol) bovine serum albuminand 0.05% TWEEN™-20 detergent prior to use. The conjugate was allowed toincubate for 30 minutes at room temperature in a moist chamber. Afterincubation, the conjugate was removed from the wells, and each well waswashed 3 times with wash buffer as described above. After the last washwas removed from the wells, 0.1 mL of alkaline phosphatase substratesolution was placed in each well, and the plate was incubated for 30minutes at room temperature in a moist chamber to allow colordevelopment. The color was read at 405 nm using a spectrophotometer.

The test absorbance values were converted to Relative ELISA Values (REV)by expressing the test serum absorbance value as a ratio of theabsorbance of the calibrator serum. This was performed as follows:

(i) obtain the absorbance value for the blank well(s) containingeverything but the serum;

(ii) obtain absorbance values for test and calibrator samples;

(iii) subtract the blank absorbance value from each of the test andcalibrator sample values to obtain corrected values;

(iv) multiply the calibrator absorbance by the calibration factor(calibration factor is the factor by which the calibrator [a lowpositive serum] absorbance must be multiplied to reach the cut-offpoint, which is three standard deviations above the mean negativeabsorbance of a population) indicated on the calibration serum to obtainthe ELISA cutoff value; and

(v) divide the corrected absorbance for each test serum by the cutoffvalue to obtain the REVs. REVs equal to or greater than 1.0 areconsidered positive for IgM antibodies against rubella virus, whilethose below 1.0 are considered negative.

Example 5 Comparative Studies of Assay Accuracy

A panel of 102 serum samples that were known negative for IgM antibodiesand 25 serum samples that were known positive for IgM antibodies wereassayed by the methods of the instant invention. The same sera wereassayed with a commercially available kit and by a preexisting in-houseELISA, both of which employed detergent-disrupted purified virus as theantigen and a serum diluent lacking urea. All three tests correctlyidentified the positive sera.

Table I shows that the commercial kit and the preexisting in-house ELISAyielded significant numbers of false positive reactions within thenegative serum panel, whereas the ELISA of the instant invention (“New”ELISA) correctly identified all 102 negative samples.

TABLE I Comparative Assays of 102 Known Negative Samples ASSAY POSITIVENEGATIVE In-house ELISA 16 86 Commercial Kit 7 95 “New” ELISA 0 102

Example 6 “New” ELISA without urea

100 negative serum samples were tested using the ELISA of the instantinvention (“New” ELISA) employing rubella E1 and E2 glycoproteins asantigen. Urea was omitted from the sample diluent. No false positiveresults were obtained, but the relative ELISA values (REVs) ranged from0 to 0.8, a fairly broad range. Samples yielding REVs of 1.0 or greater(as discussed above) are considered positive for the presence ofanti-rubella IgM antibodies. However, approximately 90% of the obtainedREVs were 0.2 or less, indicating that use of the E1 and E2glycoproteins as antigen in immunoassays is very effective in theelimination of false positive results even when no urea is added to thesample diluent.

Example 7 “New” ELISA with urea

The same 100 negative serum samples were tested using the ELISA of theinstant invention (“New” ELISA) employing E1 and E2 glycoprotein asantigen and further employing a sample diluent containing 3 M urea. Nofalse positive results were obtained, and furthermore, the REVdistribution was narrowed to a range of 0 to 0.4, with over 95% of thevalues equal to 0. This indicates that the “New” ELISA using both the E1and E2 glycoprotein antigens and urea in the sample diluent is superiorin sensitivity to the ELISA using only the E1 and E2 glycoproteinantigens.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spirit,and scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention.

What is claimed is:
 1. A method for detecting anti-rubella IgMantibodies, comprising: a) providing a test sample suspected ofcontaining anti-rubella IgM antibodies; b) contacting the test samplewith rubella antigens comprising rubella E1 glycoprotein and rubella E2glycoprotein and substantially free of rubella capsid protein to form abinary complex, wherein the E1 and E2 glycoproteins are purified fromrubella virus and wherein said binary complex is a complex of rubellaantigens and IgM; c) contacting the binary complex with an indicatorreagent to form a ternary complex; and d) detecting the presence orabsence of the ternary complex; wherein said method specifically detectsanti-rubella IgM antibodies and substantially eliminates false positiveresults.
 2. The method of claim 1, wherein the test sample is obtainedfrom a bodily fluid.
 3. The method of claim 1, wherein the test sampleis obtained from blood.
 4. The method of claim 1, wherein the testsample is diluted with a diluent comprising urea prior to contacting thesample with the rubella antigens.
 5. The method of claim 4, wherein thediluent comprises between about 2 M urea and about 4 M urea.
 6. Themethod of claim 4, wherein the diluent comprises about 3 M urea.
 7. Themethod of claim 1, further comprising washing the binary complex orternary complex with a wash comprising urea.
 8. The method of claim 7,wherein the wash comprises between about 2 M urea and about 4 M urea. 9.The method of claim 1, wherein the rubella antigens are immobilized on asolid support.
 10. The method of claim 9, wherein the solid support is amembrane, filter, piece of plastic, piece of glass, or bead.
 11. Themethod of claim 9, wherein the solid support is polypropylene,polystyrene, polyvinyl chloride, polyamide, polycarbonate, polyether,polymethyl methacrylate, nitrocellulose, polyvinylidene difluoride,agarose, metal, or nylon.
 12. The method of claim 1, wherein theindicator reagent is conjugated to a detectable label.
 13. The method ofclaim 12, wherein the detectable label is a protein, enzyme,radioisotope, nucleic acid segment, or fluorochrome.
 14. The method ofclaim 13, wherein the enzyme is horseradish peroxidase, alkalinephosphatase, or β-galactosidase.
 15. The method of claim 13, wherein theenzyme catalyzes the conversion of a non-chemiluminescent reagent into achemiluminescent product.
 16. The method of claim 13, wherein the enzymecatalyzes the conversion of a non-colorimetric reagent to a colorimetricproduct.